Single-cell transcriptomics reveal cellular diversity of aortic valve and the immunomodulation by PPARγ during hyperlipidemia

Valvular inflammation triggered by hyperlipidemia has been considered as an important initial process of aortic valve disease; however, cellular and molecular evidence remains unclear. Here, we assess the relationship between plasma lipids and valvular inflammation, and identify association of low-density lipoprotein with increased valvular lipid and macrophage accumulation. Single-cell RNA sequencing analysis reveals the cellular heterogeneity of leukocytes, valvular interstitial cells, and valvular endothelial cells, and their phenotypic changes during hyperlipidemia leading to recruitment of monocyte-derived MHC-IIhi macrophages. Interestingly, we find activated PPARγ pathway in Cd36+ valvular endothelial cells increased in hyperlipidemic mice, and the conservation of PPARγ activation in non-calcified human aortic valves. While the PPARγ inhibition promotes inflammation, PPARγ activation using pioglitazone reduces valvular inflammation in hyperlipidemic mice. These results show that low-density lipoprotein is the main lipoprotein accumulated in the aortic valve during hyperlipidemia, leading to early-stage aortic valve disease, and PPARγ activation protects the aortic valve against inflammation.

The study is well designed and comprehensive. It provides some mechanistic insight in aortic valvular inflammation and thus improves our knowledge on the pathophysiology of aortic valve disease and may offer potential therapeutics target. Nevertheless, some points should be addressed to further strengthen the conclusions of this study.
1. In figure 1, the authors analyzed lipid accumulation in blood and aortic valves in WT, Apoe-/-and Ldlr-/-mice. Despite similar plasma levels of total cholesterol and LDL in Apoe-/-and Ldlr-/-, Ldlr-/-showed increased accumulation of lipids and LDL in aortic valve. What about macrophage accumulation, is it also different in aortic valve from Apoe-/-and Ldlr-/-mice? This should be addressed since the authors use this parameter to evaluate inflammation. Explanation for the difference in LDL accumulation in aortic valves between Apoe-/-and Ldrl-/-mice should be provided. Is this decreased in lipid accumulation in aortic valve from Apoe-/-mice explained by differences in expression of LDL scavenger receptors. It is not clear which mice were used in Fig1h and j, the information is missing from the result and figure legend.
2. The authors showed that increasing plasma LDL levels in Apoe-/-mice enhanced lipid and macrophage accumulation in aortic valves. To further support this causal relationship between lipid accumulation and aortic valvular inflammation, the authors should consider analyzing the effect of reducing LDL levels in Ldlr-/-mice on aortic valve inflammation. 3. Single cell RNA sequencing analysis revealed heterogeneity in VIC, VEC and leukocytes. VICs show proinflammatory features. What about the VECs?. Among the VEC subpopulations, one express Prox-1. Are these cells lymphatic endothelial cells? 4. Single cell RNA sequencing analysis also reveal the activation of PPAR-g pathway in VECs. Is this also observed in VICs and macrophages? 5. The staining of PPAR is not obvious and costaining with a marker of VECs should be performed in Fig  7e. For this experiment, the authors used section from Apoe-/-which showed less lipid accumulation and inflammation. What about in Ldlr-/-mice? To confirm the activation of PPAR in VECs, the cytoplasmic and nuclear cellular localization of PPAR should be analyzed and/or the analysis of PPAR targeted genes should be examined. This should also be performed for VIC and macrophages since this pathway is also activated in these cells based on the single cell RNA sequencing.
6. The authors propose that PPAR pathway regulates the recruitment of monocyte into the valve. Do the activation or inhibition of PPAR also affect the frequency and number of circulating blood monocyte which are known to be modulated in hyperlipidemic mice. Can the authors pinpoint to candidate gene/protein regulated by PPARexpressed by pro-inflammatory VECs involved in the control of monocyte recruitment? Such as chemokine, adhesion molecules.
In this paper, the authors characterize the cellular content of valvular inflammation under hyperlipidemia. They confirm an association between increased plasma LDL levels and increased valvular lipids and macrophage inflammation. They performed scRNAseq analysis to study the cellular heterogeneity of aortic valves under hyperlipidemia in 2 mouse models: ApoE and LDLR knockout. They confirm that PPAR pathway activation reduces inflammation thus putting this pathway forward as a potential drug target for aortic valve disease.
The introduction gives a good and generalized background of the topic and clearly states the motivations to undergo this research. The methods are well developed and explained and provide enough details to allow reproducibility, they also comply with the requested standards in the field. The results are well presented, analyzed, and interpreted. However, the discussion lacks in-depth analysis.
In the current state, the results presented in this paper lack originality and do not bring a major contribution to the field without further investigation. Of note, the scRNAseq analysis is the most noteworthy information but it has not been taken deep enough to provide compelling and significant contributions to the field. Despite a lack of novelty, a good and reliable amount of work leading to convincing results has been performed. It would be unfortunate not to take advantage of the author's expertise to further decipher the mechanisms involved in this disease affecting a lot of individuals.
Additional comments: • It would be instrumental in adding ApoE-/-and LDLR-/-without high-fat diet as controls.
• Regarding the monocyte migration assay, LDL and ox-LDL alone (without interstitial cells) should be tested.
• In supplementary Fig. 3, it is mentioned that Clec3b+ is not located within the aortic valves. Does it mean that there is possible contamination by aortic cells?
• To strengthen the scRNAseq results and PPARγpathway implication (as mentioned in the discussion) specific deletion of PPARγin vascular endothelial cells is mandatory and would provide the novelty lacking in this paper.
Reviewer #4 (Remarks to the Author): Manuscript NCOOMS-21-49026 Single-cell transcriptomics reveal cellular diversity in the aortic valve and immunomodulatory role of PPARg during hyperlipemia The authors demonstrated that lipid accumulation and inflammation in the aortic valve is triggered by LDL in different hyperlipidemic mouse models (WD fed Ldlr-/-and Apoe-/-). They performed a comprehensive scRNAseq analysis of diseased vs. normal aortic valves that revealed two main Mo/Ma populations (monocyte-derived population with pro-inflammatory profile and resident Lyve+ with antiinflammatory profile. They compared the kinetics of lipid deposits, inflammation, and monocyte/macrophage infiltration in hyperlipidemic mouse models with different genetic manipulation. The manuscript is well written, and the study is very well designed. The introduction is concise and covers all the topics developed in the paper. However, the novelty of the study is not very clear. The authors describe the accumulation of lipids, in particular LDL, and macrophages in the aortic valve of well-studied and standardized mouse models of hyperlipidemia and atherosclerosis. Comparing two different genetic deletions (Ldl-/-and Apoe-/-) for the development of aortic valve disease might not provide sufficient insight regarding the mechanisms of the disease and how lipid-lowering drugs could be useful in preventing the initiation of the disease. It might be worthwhile to compare either WD-fed Apoe-/-and Ldl-/-with chow-diet fed Apoe-/-and Ldlr-/-mice to gain insight in the diet role in the development of the disease or compare WD-Apoe-/-and Ldlr-/-with WD-C67BL6 mice to elucidate genetic background role. The findings that the PPARg pathway is activated in hyperlipidemic mice and the corroboration of these findings in human samples make PPARg a possible target to be further evaluated for aortic valve disease. I recommend the authors restructure the figures to better emphasize these findings.
2) Please add statistical analysis to each figure legend and the times that the independent experiment has been performed.
3) According to animal experiments section in M&M, the PCSK9-AAV injection was also performed in WT mice, please describe the findings in  Fig. 6E to confirm the increase in CD36+ VEC cells in diseased Ldlr-/-valve compared to normal C57BL6 valves. 7) Please quantify in situ hybridization experiment in Fig. 7e to state "The valves from aortic hyperlipidemic mice showed much higher PPARg expression in nuclei, especially (in particular) in VECS, compared to normal valves (page 16). Fig. 7, the authors describe that PPARg pathway in VECs is activated by increased LDL plasma levels, although LDL and cholesterol levels are only increased (above the reference values for humans) in 2 patients. Could the authors comment on this correlation and the fact that PPARg+ is highly increased in non-calcified vs. calcified lesion?

Reviewer #1
This manuscript describes the regulation of valvular disease in mice models. Using sophisticated analysis of cellular transcriptomes, the authors report that a detailed analysis of changes in compositions of the valve during disease onset. Among the regulated genes, the PPARg pathway was predominantly regulated in one cluster of VEC of diseased mice. While inhibition of PPARg further increased valve inflammation, pharmacological activation of PPARg reduced valve disease. The study comprises extensive data and is generally well performed. However, the findings are not very surprising and the role of PPARs in valve disease has been previously reported (see review www.ncbi.nlm.nih.gov/pmc/articles/PMC6719701/).
-Thank you so much for your valuable comments. As you pointed out, our study is in line with previous reports that support the role of PPARG in aortic valve disease. However, previous reports have dealt with late-stage aortic valve disease (involving calcification). In contrast, our study deals with the early stage of aortic valve disease. We showed a protective role of PPARγ in the early stage of aortic valve disease, via scRNA-seq. In the revised manuscript, we further suggested that the anti-inflammatory effect of PPARγ is also exerted in a VEC-specific manner, by RNA-seq analysis, using PPARG gene silencing of human aortic VEC (Figure 7f-g) [page 19, lines 385-392]. We believe that the improvement in our study's content will address the issue of novelty, and hope our revised manuscript and the point-by-point responses will fulfill your expectations.  Figure 6e, the authors showed a representative image of increased CD36 expression. However, these data are not quantified. An analysis of CD36 in early and later stages of the disease should be done to determine if the induction is indeed an early protective mechanism, which may be lost at later stages of the disease.
-We appreciate your valuable comment. As requested, we have added the quantification of Cd36 in situ hybridization (Figure 6f). Owing to difficulties in obtaining early stenosis samples in humans, or late samples in mice, directly comparing early and late stenosis data is difficult. We would like to emphasize that Syvaranta et al. reported two observations of CD36 expression 1 . First, valve tissues expressed lower levels of CD36 in stenosis than in controls. Second, myofibroblasts isolated from late-stage stenosis expressed higher levels of CD36 in culture than in those isolated from controls. We also checked the previously reported scRNA-seq data by Xu et al. 2 , and found that the calcified group expressed higher levels of CD36 than the non-calcified group (Extra figure 1). Unlike previous reports, our experiments focused on early-stage disease during which lipid accumulation and inflammation mainly occur. We compared the aortic valves of hyperlipidemic mice with those of normal mice, and found that CD36 was upregulated in aortic valve hyperlipidemic conditions (Figure 6f). Upregulated CD36 may play a role in valvular lipid accumulation in early-stage disease, supported by the decrease in LDL accumulation in the aortic valve by CD36 antagonist (salvianolic acid B; SAB) treatment ( Figure 1j). Overall, these observations suggest that different stimuli can cause complex effects on CD36 expression. It is likely that cell populations in early-and late-stage stenosis are exposed to different levels of external stimuli. Molecular alterations during the progression of stenosis require further investigation. Fig. 6f. Localization of Cd36 + VEC in the aortic valve with quantification.

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Identification of the localization of Cd36 + VEC subclusters using RNA in situ hybridization. The graph indicates the quantification of the in situ hybridization using the CD36 probe (n = 5). Scale bar: 50 μm.  2. Please provide more controls for the single cell seq data sets (e.g. number of reads, mapped reads, genes/cell, UMI/cell, total genes per sample) to make sure that the quality is similar among all studied samples.
-The requested information is provided in Supplementary Table 3 ("Summary of scRNA sequencing parameters").  -Thank you for your helpful review of our work. Thanks to your crucial and detailed comments, we performed additional experiments and revised the manuscript accordingly. We have attempted to answer as many points as possible, and hope our revised manuscript and the point-by-point responses will fulfill your expectations.

3-2. Among the VEC subpopulations, one express Prox-1. Are these cells lymphatic endothelial cells?
-VEC_C2 (Prox1 + EC) cells were localized in the endothelial lining of the fibrosa, and they did not form structures within lymphatic vessels (Figure 6f). Except for the localization displayed in Figure 6f, none of the other regions had PROX1 + VECs. Lymphatic endothelial cells expressed Lyve1 as a marker gene 3, 4 , but Lyve1 was not expressed in VEC_C2 (Prox1 + EC) cells (Extra figure 2). In this regard, we conclude that VEC_C2 (Prox1 + EC) cells are not a type of lymphatic EC. Fig. 6f. Localization of PROX1 + VEC in aortic side of the aortic valve. Scale bar: 50 μm.  Immunostaining of PPARγ (red) and endomucin (EMCN, EC marker, green) in aortic valve with sinus from normal (chow diet) and hyperlipidemic mice (Apoe -/and Ldlr -/mice, WD for 16 weeks) (n = 4). DAPI (blue) was used to stain nuclei. The graph represents the mean fluorescence intensity (MFI) of PPARγ in VECs. Kruskal-Wallis test with post-hoc Dunn's test was used. Scale bar: 30 μm.

5-2. To confirm the activation of PPARγ in VECs, the cytoplasmic and nuclear cellular localization of PPARγ should be analyzed and/or the analysis of PPARγ targeted genes
should be examined. This should also be performed for VIC and macrophages since this pathway is also activated in these cells based on the single cell RNA sequencing.
-To confirm the activation of PPARγ in VECs, we analyzed the relative expression of PPARγ targeted genes (the gene list from PPARgene database) 5   -We fed pioglitazone-WD (experimental group) or normal WD (control group) to C57BL/6J mice injected with PCSK9-AAV, using the same method as that shown in Figure   8d. We compared the number of blood monocytes and subsets (Ly6C-high/low), for 6 weeks, by flow cytometric analyses once a week (Supplementary Figure 15c-d). No difference in the number of monocytes or subsets was found at each of the checkpoints. We present these data in Supplementary Figure 15c Flow cytometry analysis of circulating blood monocytes, for 6 weeks. PCSK9-AAV-injected C57BL/6J mice were fed with pioglitazone-containing WD or normal WD for 6 weeks, and the number of monocytes and subsets in 10 μL of blood was examined once a week (n = 5). c. The gating strategy of blood monocytes and subsets. d. Cell counts of monocytes, Ly6C high monocytes, and Ly6C low monocyte in 10 μL of blood, from week 1 to 6.     (Figure 1a-d and Figure 2a-c). These results may be derived from differences in the blood lipid profiles of mice. Unlike that which was observed in hyperlipidemic models (Apoe -/and Ldlr -/mice), feeding WD to wild-type (C57BL/6J) mice did not change blood total cholesterol and LDL levels (Figure 1b and Supplementary Figure 5b

Regarding the monocyte migration assay, LDL and ox-LDL alone (without interstitial cells) should be tested.
-We added data for non-treated, LDL, and oxLDL-alone (without VICs) treated mice to Figure 5J. These data were obtained simultaneously with pre-existing data (with VICs), within the same experimental batch, but were not included in the initially submitted manuscript.

In supplementary Fig. 3, it is mentioned that Clec3b+ is not located within the aortic valves. Does it mean that there is possible contamination by aortic cells?
-First, based on our anatomical criteria (Supplementary Figure 1b), we isolated the aortic valve leaflet only; therefore, the possibility of aortic cell contamination was very low.
If contamination had occurred, smooth muscle cells (SMCs), the main cellular components of the aortic wall, should have been present as much as VICs. However, in our scRNA-seq data, SMCs were not a major population. Nevertheless, we considered the previous Supplementary

To strengthen the scRNAseq results and PPARγ pathway implication (as mentioned in the discussion) specific deletion of PPARγ in vascular endothelial cells is mandatory
and would provide the novelty lacking in this paper.
-We appreciate your helpful comment. To reinforce our hypothesis, we performed new bulk RNA-seq using human aortic valvular endothelial cells that were isolated from patients who underwent aortic valve replacement and had been cultured with oxLDL treatment and siRNA-based PPARG knockdown (Figure 7f-g, Supplementary Figure 14, and Supplementary Data 4). Upon treatment with oxLDL, knockdown of PPARG in VECs induced upregulation of pro-inflammatory genes, such as CXCL1, CCL2, CXCL16, and IL6, and cell adhesion molecules, such as ICAM1 and ICAM2 (Figure 7f-g). We have added this data to Figure 7f-g, Supplementary Figure 14

The manuscript is well written, and the study is very well designed. The introduction is
concise and covers all the topics developed in the paper. However, the novelty of the study is not very clear.
-Thank you so much for your valuable comments. In the revised manuscript, we have added new data, such as RNA-seq analysis using PPARG gene knockdown of human aortic VECs (Figure 7f-g). Therefore, we have further supported our findings and improved the premise of the manuscript. We believe that our revised manuscript highlights the novelty of the study, and hope our revised manuscript and the point-by-point responses will fulfill your expectations.
.  (Figure 1a-d, Figure 2a-c, and   Supplementary Figure 5a-c). In addition, the fold change in WD-fed mice versus chow-fed mice was much higher in Ldlr -/than in Apoe -/mice, which corresponds with our previous analysis that revealed that Ldlr -/mice had a higher lipid accumulation and larger portion of valvular immune cells (especially MHC-II hi macrophages) than did Apoe -/mice among WDfed mice. This observation may be caused by a higher blood LDL level in Ldlr -/mice than in Apoe -/mice (Figure 1a-d, Figure 2a (Figure 1a-d, Figure 2a    -Thank you for your suggestion. In the revised manuscript, we have added new data and reconstructed the figures to strengthen our statement of the protective role of PPARγ in aortic VECs, in a hyperlipidemic condition (Figure 7f-g, Supplementary Figure 14 and   12c-d) [page 19, lines 385-392]. We believe that these newly included data will adequately highlight our main findings. -We changed 'infected' into 'injected' in the legends of Figure 2 and 8. 2) Please add statistical analysis to each figure legend and the times that the independent experiment has been performed.

I-1: The authors describe the accumulation of lipids, in particular LDL
-We have added an explanation of the statistics as well as the information on the number of times for each independent experiment to each figure legend.

3) According to animal experiments section in M&M, the PCSK9-AAV injection was
also performed in WT mice, please describe the findings in Figure 2.
-Confusion may occur from integration of the methods of two different experiments, using PCSK9-AAV in the "Mice" section. In the experiment in Figure 2d-i, PCSK9-AAVinjected Apoe -/mice were used in the experimental group, and non-injected Apoe -/mice were used in the control group. C57BL/6J (wild-type) mice were not used in this experiment.
C57BL/6J (wildtype, Ccr2 +/+ ) mice were used for the experiment Figure 3f Fig 4 (i.e. 16 weeks) -We have inscribed the time point in the legend of Figure 4. Fig. 6E to confirm the increase in CD36+ VEC cells in diseased Ldlr-/-valve compared to normal C57BL6 valves.

6) Please quantify in situ hybridization experiment in
-We have quantified RNA in situ hybridization of Cd36 (Figure 6f). Fig. 6f. Localization of Cd36 + VEC in the aortic valve with quantification.

Part of
Identification of the localization of Cd36 + VEC subclusters using RNA in situ hybridization. The graph indicates the quantification of the in situ hybridization using the CD36 probe (n = 5). Scale bar: 50 μm. Fig. 7e Fig. 13. Immunostaining of PPARγ in mouse aortic valve.

7) Please quantify in situ hybridization experiment in
Immunostaining of PPARγ (red) and endomucin (EMCN, EC marker, green) in aortic valve with sinus from normal (chow diet) and hyperlipidemic mice (Apoe -/and Ldlr -/mice, WD for 16 weeks) (n = 4). DAPI (blue) was used to stain nuclei. The graph represents the mean fluorescence intensity (MFI) of PPARγ in VECs. Kruskal-Wallis test with post-hoc Dunn's test was used. Scale bar: 30 μm. Fig. 7, the authors describe that PPARg pathway in VECs is activated by increased LDL plasma levels, although LDL and cholesterol levels are only increased (above the reference values for humans) in 2 patients. Could the authors comment on this correlation and the fact that PPARg+ is highly increased in non-calcified vs. calcified lesion?

8) In
-Thank you for your comments. As you mentioned, only two patients in the noncalcified group showed hypercholesterolemia (total cholesterol > 200 mg/dL; Supplementary Table 1). However, PPARγ + VECs showed positive correlations with both total cholesterol and LDL levels (Figure 7j), which may be due to the influence of blood total cholesterol and LDL levels on PPARγ activity -even under conditions of normal cholesterol levels. This may be explained by mouse aortic valve scRNA-seq analyses (Supplementary Figure 12c).
Overall, VECs showed higher expression of PPARγ target genes than did VICs or macrophages, not only in hyperlipidemic (Apoe -/and Ldlr -/-, WD-fed) mice, but also in normal controls (C57BL/6J, chow-fed; Supplementary Figure 12c). It seems that, to some extent, PPARγ is basally activated in VECs, and this activation may be enhanced as blood total cholesterol and LDL levels increase. We think this tendency seems to be conserved in humans.
In our IHC results, PPARγ + valvular cells and PPARγ + VECs were higher in noncalcified lesions than in calcified lesions (Figure 7h-   Single-cell transcriptomics reveal cellular diversity in the oartic valve and the immunomodulatory role of PPARg during hyperlipidemia The manuscript describe the heterogeneity of cells in the early aortic valve disease and empathize the role of PPARg as a key anti-inflammatory regulator in the pre-calcified stage of aortic valve disease. The authors have effectively clarified all the suggestions and concerns from Revision 1 providing the a better background in premise of the manuscript. Overall, the study is well design and comprehensive, providing mechanistic insights of early aortic valve disease under hyperlipidemic condition and scRNAseq and FACS, IHC complementary data has shown possible pathways to be targeted as therapeutic options. The methodology is appropriate and meets the expected standards in the field, providing enough details for the work to be reproduced. Largely, the manuscript has a reliable number of results backing the conclusions of the study. Nevertheless, other points should be addressed to strengthen the manuscript. Minor comments that remain to be addressed: 1) Please add statistical analysis of Ldlr-/-vs. Apoe-/-in Fig. 1b to claim "LDL, but not total cholesterol, was higher in Ldlr-/-than in Apoe-/-mice".
2) Please clarify the scientific reason of why the PCSK9 AAV injection experiment was only performed in Apoe-/-mice and, in the next figure ezetimibe lipid-lowering treatment was only performed in LDLr-/but not in Apoe-/-mice. Consistently evaluating both LDLr-/-and Apoe-/-backgrounds in the manuscript will provide more strength to the conclusions of the study.
3) Supplementary data 1: please provide insights in the cellular pathways that suggest a phenotypic and functional heterogeneity in macrophages subclusters. Although the results are shown in Supplementary Data 1, the description of Sup. Data 1 is lacking in the results section. 4) Lastly, PPARg agonist + lipid lowering treatment combination could provide a more suitable treatment for early aortic valve disease? Can the authors speculate or test synergism between these two treatments in decreasing lipid accumulation and leukocyte infiltration or inflammation and aortic valve disease progression?
Overall, the authors have extensively discussed and addressed all valuable comments from the reviewers in the revised manuscript. The addition of siRNA-based PPARg knockdown in vitro experiment has provide a valuable mechanism insight in human disease. The authors could discuss about the therapeutic perspectives of the modulation of PPARg pathway and associate with different vascular diseases and disease stage. In the current state, the results are adequate and present a comprehensive characterization of valvular inflammation and hyperlipidemia. However, addressing minor comments would definitely enhance the strength of the current version.

NCOMMS-21-49026 2 nd revision
Point-by-point response Reviewer #1 N/À -We really appreciate your positive review of our revised manuscript.

Reviewer #2
The authors have addressed all the reviewer's comments. The revised manuscript has been improved. No further comments.
-We really appreciate your positive review of our revised manuscript.

Reviewer #3
In this revised paper, the authors performed convincing additional experiments, addressing my comments. Despite well-designed experiments and cutting-edge methodology, the authors did not properly extract the novelty of their results from the current literature in the field. In particular, it appears that their results are mostly related to early stages of aortic valve disease. This needs to be stressed more in the discussion and highlighted in the abstract and title.
-Thank you so much for your valuable comments. As you pointed out, our study is mainly about the very early stage of aortic valve disease having local inflammation, and we agree that it is the crucial novelty of the study. Thus, we revised the statements of abstract in the 2 nd revised version of the manuscript, to better highlight the novelty of our study.

Minor comments:
• In Figure 1a, in C5BL/6J mice Western diet, it seems that the oil-red-o staining is strong in the valves, which are not highlighted. Please clarify.
-In C57BL/6J-WD of Fig. 1a, we can see black (or dark brown) pigments in the valve leaflet. These are the melanin pigments of aortic valves. There are plenty of melanin pigments in the mouse aortic valve, as shown in the left panels of Supplementary Fig. 1a-b. Surely, the valvular oil red o positive area of C57BL/6J mice was extremely low in both chowfed and WD-fed groups. We added arrowheads in Fig. 1a indicating accumulated lipids (oil red o positive area) and the following statement in the figure legend to avoid confusion: Black or dark brown spots are melanin pigments.  -In our first revision, we showed the CD36 expression between calcified and noncalcified human samples as Extra fig. 1 to respond to the comments of reviewer #1. In total valvular cells and VICs, it seemed that expression of CD36 was relatively higher in calcified compared to non-calcified, but as shown in the feature plot, the overall expression level of CD36 was extremely low (Supplementary Fig. 14). Now we presented these data (previous Extra fig. 1) as the new supplementary figure (Supplementary Fig. 14)  The methodology is appropriate and meets the expected standards in the field, providing enough details for the work to be reproduced. Largely, the manuscript has a reliable number of results backing the conclusions of the study. Nevertheless, other points should be addressed to strengthen the manuscript.
-Thank you so much for your helpful comments. We faithfully responded to all comments as much as possible, and hope our responses will suffice to answer the questions.
Minor comments that remain to be addressed: Fig. 1b to claim "LDL, but not total cholesterol, was higher in Ldlr-/-than in poe-/-mice".

1) Please add statistical analysis of Ldlr-/-vs. poe-/-in
-We added the statistical analysis of Ldlr -/-WD versus Apoe -/-WD for total cholesterol and LDL level in Fig. 1b.   Fig. 1a-d  -The molecular mechanism of PCSK9 is binding and preventing LDLR recycling -Thanks to the reviewer's valuable comments, we were able to strengthen the context of the study. Once again, we appreciate your dedication to reviewing our manuscript.